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Master's Thesis Template UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE CHARACTERIZATION OF FUNGAL CONTAMINANTS IN B20 BIODIESEL STORAGE TANKS AND THEIR EFFECT ON FUEL COMPOSITION A THESIS SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of MASTER OF SCIENCE By ODERAY ANDRADE ELJURI Norman, Oklahoma 2016 CHARACTERIZATION OF FUNGAL CONTAMINANTS IN B20 BIODIESEL STORAGE TANKS AND THEIR EFFECT ON FUEL COMPOSITION A THESIS APPROVED FOR THE DEPARTMENT OF MICROBIOLOGY AND PLANT BIOLOGY BY ______________________________ Dr. Bradley Stevenson, Chair ______________________________ Dr. Amy Callaghan ______________________________ Dr. Boris Wawrik © Copyright by ODERAY ANDRADE ELJURI 2016 All Rights Reserved. “Science is more than a body of knowledge. It is a way of thinking; a way of skeptically interrogating the universe with a fine understanding of human fallibility.” -Carl Sagan To Matias. You are my candle in the dark. To Sebastian and Adrian. My biggest dream is to be part of your adventures and accomplishments, as you are being part of mine. Acknowledgements First, I would like to thank the Secretariat for Higher Education, Science, Technology and Innovation of the Republic of Ecuador “SENESCYT”, for granting me with a scholarship to study abroad. I would like to express my gratitude to my advisor, Dr. Bradley S. Stevenson. Thank you for giving me the opportunity to be part of the Stevenson Research Group. Your office door was always open when my questions and doubts arose. Your love for Microbiology is an inspiration. I would like to acknowledge my laboratory colleagues: Blake Stamps, Brian Bill, Heather Nunn and James Floyd. Our lives crossed paths in this fascinating journey through Microbiology, and from you all I learned how the ‘circle of niceness’ works. Thank you Blake, for the fuel collection and support on the isolation of the microorganisms and fuel characterization. James, thank you for the assistance in the preparation and viewing of scanning microscope images. Heather, thank you for all of your advice on developing my experimental designs. Brian, I am indebted to your valuable comments and constant feedback during the writing process of this thesis. Special thanks goes to Matias Robayo, for his tireless and skillful collaboration in the statistical analysis. Funding for this project was provided by the COP Technical Corrosion Collaboration grant #FA7000-15-2-0001. iv Table of Contents Acknowledgements ......................................................................................................... iv List of Tables .................................................................................................................. vii List of Figures ................................................................................................................ viii Abstract ............................................................................................................................. x Chapter 1: Problem Statement .......................................................................................... 1 Chapter 2: Characterization of Fungal Contaminants from B20 Storage Tanks .............. 9 2.1 Introduction .......................................................................................................... 9 2.2 Materials and Methods ....................................................................................... 14 2.2.1 Sample Collection and Cultivation ............................................................ 14 2.2.2 Molecular Identification of Fungal Isolates .............................................. 15 2.2.3 Phenotypic and Biochemical Characterization .......................................... 17 2.2.4 Chemotaxonomic Characterization ........................................................... 19 2.2.5 B20 Biodegradation Experiments with Fungal Isolates ............................ 19 2.3 Results ................................................................................................................ 21 2.3.1 Isolation ..................................................................................................... 21 2.3.2 Characterization of the Filamentous Fungus Byssochlamys (strain ID: SW2) ....................................................................................................... 21 2.3.3 Characterization of the Yeast Wickerhamomyces (strain ID: SE3) .......... 22 2.3.4 Fungal Biodegradation of B20 biodiesel ................................................... 23 2.4 Discussion ........................................................................................................... 26 Chapter 3: Chemical Analysis of B20 Biodiesel Fuel Exposed to Contaminated Underground Storage Tanks and its Correlation to Fungal Biodegradation ...... 49 v 3.1 Introduction ........................................................................................................ 49 3.2 Materials and Methods ....................................................................................... 52 3.2.1 Sampling and GC/MS data collection ....................................................... 52 3.2.2 Chromatographic Data Analysis ................................................................ 53 3.2.3 Fungal Biodegradation Classification Model ............................................ 54 3.3 Results ................................................................................................................ 56 3.3.1 Characterization of the Composition of B20 Fuel Samples ...................... 56 3.3.2 Fungal Biodegradation Model ................................................................... 57 3.4 Discussion ........................................................................................................... 59 Chapter 4: Summary and Future Directions ................................................................... 73 References ...................................................................................................................... 77 vi List of Tables Table 2.1 Identity and provenance of isolated fungi. ..................................................... 34 Table 2.2 Physiological characteristics of Wickerhamomyces anomalus SE3. .............. 38 Table 2.3 Physiological characteristicsa of Byssochlamys sp. SW2 ............................... 39 Table 3.1 Common fatty acid methyl esters found in biodiesel a. .................................. 64 Table 3.2 Retention times of major peaks identified in B20 fuel samples. .................... 66 Table 3.3 Principal component analyses (PCAs) of global B20 fuel dataset. ................ 67 Table 3.4 Description of the test set of B20 samples obtained from SE facility and used to validate the LDA model. ............................................................................................ 71 vii List of Figures Figure 1.1 Transesterification reaction scheme to produce biodiesel .............................. 3 Figure 1.2 Images of B20 fuel samples obtained from a storage tank at a USAF facility 5 Figure 1.3 Fungal communities detected at the bottom of storage tanks ......................... 6 Figure 2.1 Morphology of Byssochlamys sp. SW2 and Wickerhamomyces anomalus SE3 ................................................................................................................................. 35 Figure 2.2 Maximum Likelihood tree based on 28S rRNA sequence phylogeny of the yeast Wickerhamomyces anomalus SE3 and its close relatives ..................................... 36 Figure 2.3 Maximum Likelihood tree based on 28S rRNA sequence phylogeny of the filamentous fungus Byssochlamys sp. SW2 and its close relatives ................................ 37 Figure 2.4 Degradation of FAME in B20 biodiesel after 7 days of incubation with isolates Wickerhamomyces anomalus SE3 and Byssochlamys sp. SW2 ........................ 40 Figure 2.5 Degradation of hydrocarbons in B20 biodiesel after 7 days of incubation with isolate Wickerhamomyces anomalus SE3 ...................................................................... 41 Figure 2.6 Growth curve for Wickerhamomyces anomalus SE3 in ASW medium containing B20 biodiesel as sole carbon source ............................................................. 42 Figure 2.7 Degradation of FAME in B20 biodiesel after 30 days of incubation with isolate Byssochlamys sp. SW2 ........................................................................................ 43 Figure 2.8 Representative total ion chromatograms of aqueous phase after 80 days of biodegradation of B20 with isolate Byssochlamys sp. SW2. ......................................... 44 Figure 2.9 Effect of temperature on growth of the yeast Wickerhamomyces anomalus SE3 ................................................................................................................................. 45 viii Figure 2.10 Effect of pH on growth of the yeast Wickerhamomyces anomalus SE3 .... 46 Figure 2.11 Effect of temperature on growth of Byssochlamys sp. SW2 ...................... 47 Figure 2.12 Effect of pH on growth of the Byssochlamys sp. SW2 .............................. 48 Figure 3.1 Representative total ion chromatogram (TIC) obtained from a B20 fuel sample ............................................................................................................................. 65 Figure 3.2 3-dimensional ordination of B20 fuel samples by principal components (PC) 1, 2 and 3. ......................................................................................................................
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